P988: MUTANT CALRETICULIN AND JAK2V617F DRIVER MUTATIONS INDUCE DISTINCT MITOTIC DEFECTS IN MYELOPROLIFERATIVE NEOPLASMS
Authors: Kristin Holl, Nicolas Chatain, Nathalie Brock, Susanne Krapp, Julian Baumeister, Steffen Koschmieder, Wolfram Antonin, Daniel Moreno-Andrés
Journal: HemaSphere (2023)

Topic: 15. Myeloproliferative neoplasms - Biology & Translational Research Background: Philadelphia chromosome-negative myeloproliferative neoplasms (Ph-neg. MPNs) can evolve to aggressive diseases such as secondary myelofibrosis and secondary acute myeloid leukemia. However, the molecular mechanisms of this phase transition towards more advanced disease states are ill-defined. We hypothesize that mitotic defects induced by MPN driver mutations could be critical for disease pathogenesis. Mitosis is tightly regulated by the crosstalk between Aurora B, CDK1-CyclinB1, and PLK1 kinases, and the protein phosphatase PP2A, as well as by the spindle assembly checkpoint (SAC). The SAC is a protein network recruited to chromosome kinetochores during mitosis ensuring proper chromatin segregation and karyotype stability. Aims: To determine how the most common Ph-neg. MPN driver mutations CALRdel52 and JAK2V617F affect mitotic homeostasis. Methods: We used live-cell imaging to analyze the mitotic changes of myeloid cells stably expressing the chromatin marker Histone 2B-mCherry and either the CALRdel52 or the JAK2V617F oncogene. Image analysis was used to quantify the dwell time of mitotic phases, the escape of nocodazole-induced mitotic arrest, and the frequency and type of chromatin segregation errors. Finally, we employed mRNAseq and quantitative immunofluorescence and protein expression analysis by Western blot to study the homeostasis of key mitotic regulators. Results: In comparison with 32DMPL empty vector (EV) cells, 32DMPL cells transduced with CALRdel52 or JAK2V617F showed significant increases of chromatin bridges, lagging chromosomes, and micronuclei upon DNA damage induction (doxorubicin, p<0.05, Fisher´s exact test) or SAC perturbation (inhibitor NMS-P715, p<0.02), suggesting SAC weakness. Similarly, while nocodazole treatment induced mitotic arrest in 32DMPL and human TF-1MPL EV control cells (257 ± 45 min and 313 ± 75 min, respectively), 32DMPL and TF-1MPL cells showed a significantly more rapid escape from this arrest when transduced with CALRdel52 (210 ± 36 min, p<0.02 and 177 ± 22 min, p<0.0001, one-way ANOVA) or JAK2V617F (186 ± 35 min, p<0.0001 and 201 ± 23.5 min, p<0.0001), which is congruent with a dysfunctional SAC. CALRdel52 and JAK2V617F mutant cells exhibited recruitment increases or decreases of several SAC factors to chromosome kinetochores, including Mad1, BubR1, CENP-E, CDC20, cyclin B and Aurora B (p<0.05, two tailed t-test). Western blot analysis indicated that the observed recruitment imbalances were not due to changes of total protein expression. In agreement, mRNA expression levels of these SAC factors in patient-derived CD34+ cells harboring JAK2 or CALR mutations were unchanged, when compared with healthy controls. However, mRNA expression of Aurora kinase B, PLK1 and PP2A catalytic and B55γ-regulatory subunits showed significant downregulation in JAK2 mutant CD34+ cells (two tailed t-test, p<0.05). Conclusion: CALRdel52 and JAK2V617F mutant cells show a stress-sensitive and error-prone mitosis, are predisposed to exit mitosis despite an unsatisfied SAC, and fail to reintegrate lagging chromosomes into the nucleus during telophase, resulting in micronuclei. Defective SAC factor recruitment to mitotic kinetochores due to lower expression of Aurora kinase B, PLK1 and PP2A may be responsible for the dysfunctional SAC. Our data suggest that CALRdel52 and JAK2V617F mutants induce chromatin segregation errors by disturbing the molecular control of mitosis, which in turn might promote cytogenetic abnormalities and inflammation and favor disease progression and phase transition from chronic to acute MPN states. Keywords: Second malignancy, Cell cycle progression, Chromosomal instability, Myeloproliferative disorder